Most human cells halt production of the enzyme telomerase shortly after differentiation and subsequently experience progressive shortening of chromosome ends, called telomeres. Telomere loss and several other changes seen during normal aging in vivo are also observed in primary human cells grown in culture, which stop dividing after approximately 50 cell cycles (called replicative senescence). Telomerase-deficient cells of the model eukaryote Saccharomyces cerevisiae (budding yeast) also exhibit telomere-shortening and senescence. The precise cause of senescence is unknown, but evidence suggests that exonuclease resection of the shortened telomeres leads to chromosome instability. This instability is characterized by chromosome loss and deletion events, as well as rearrangements leading to dicentric fusions and other aberrations. Aim 1: A new, regulatable telomerase expression system will be used to test proposed mechanisms of cell senescence. The lethal chromosome rearrangement model will be critically tested by asking if cells that stop growing in late senescence can be rescued by reactivation of telomerase. Experiments will also analyze mutants defective in (a) kinetochore functions that stabilize dicentrics, (b) DNA damage checkpoint responses, and (c) exonuclease-resection of telomeres. Frequencies of chromosome aberrations and telomere lengths in the rescued cells will be assessed. Aim 2: The ability of normal, checkpoint-, and resection-defective haploid cells at "the brink of death" to be rescued by mating to undamaged cells, forming diploids with shortened chromosomes complemented by good copies, will be assayed. Frequencies of aberrations in the stabilized diploids will be monitored. Aim 3: The impact of well-characterized chemical pro-oxidants and antioxidants on senescence will be determined. Survival and telomere integrity will also be monitored in cells in which genes required for resistance to oxidative DNA damage have been inactivated. Aim 4: A new approach to characterization of senescence bypass mechanisms will be employed that involves identification of high copy suppressors of in vitro cell aging. Chromosome shortening and many other metabolic changes occur during both normal human aging and also in cells grown in culture. The proposed experiments will identify genetic and environmental factors that modulate cellular aging that may also function in vivo. Chromosomes become progressively shortened in cells of most human tissues during aging, with subsequent DNA instability linked to increases in cancer and age-associated diseases. The proposed research will enhance our understanding of events that occur as a consequence of shortened chromosomes and factors that affect the rate of shortening. [unreadable] [unreadable] [unreadable]